This application claims priority to international patent application Ser. No. PCT/AU98/000189, filed Mar. 20, 1998, and to Australian patent application PO 5736, filed Mar. 20, 1997.
This invention relates to methods for detection of cancer, and in particular to the detection of endometrial cancer. The method of the invention is amenable to large-scale screening, for example in women at increased risk of endometrial cancer.
It is now clear that in many malignancies there is a high correlation between tumour aggressiveness and protease expression by the tumour cells. In the last seven years, for example, convincing evidence has accumulated that directly implicates members of the matrix metalloproteinase family in tumour invasion and metastasis.
Clinicians have long been aware of the need for developing objective criteria for the early detection of cancer, and for assessing prognostic factors when treating patients who have a malignant tumour. In general the stage of endometrial cancer, the degree of invasion into the myometrium, the histological grade and type of tumour, and the degree of invasion of the lymph-vascular space have some predictive value as to the aggressiveness of the disease. However, none of these factors is accurate, and all depend on subjective assessment. For example the type and grade of tumour are determined by histological examination, and it has been reported that assessment of these may result in a disagreement figure of up to 85% between individual different pathologists (Baak and Oort, 1991). Thus currently there is no means of predicting the prognosis for an individual patient.
It is becoming clear that the accurate prediction of the course of endometrial cancer, and the probability of survival following various types of treatment, also depend on the intrinsic properties of the malignant cells and their interaction with the host (Baak, 1991). A major objective of research in this field, therefore, is to discover the biological properties that determine the tumour""s invasive and metastatic potential.
It is known from studies with other types of cancer that quantitative techniques related to tumour cell biology, such as DNA ploidy (Iversen, 1986; van der Putten et al, 1989; Berchuck et al, 1992; Podratz et al, 1993; Lukes et al, 1994), expression of E-cadherin which mediates cell-cell adhesion (Sakuragi et al, 1994), and the secretion of matrix metalloproteinases (MMPs) involved in cell invasion (Liotta et al, 1986; Stetler-Sevenson et al, 1993) can improve the assessment of prognosis and the prediction of survival, by providing reproducible objective measures related to the behaviour of the neoplastic cells. Alterations in cellular DNA content (ploidy) have been shown to correlate with biological behaviour in cancers of the ovary, breast, colon, lung and prostate (Seckinger et al, 1989), as well as in uterine malignancies (Moberger et al, 1985; van der Putten et al, 1989). In general, cancers with a normal diploid DNA content have a better prognosis than cancers with an abnormal, aneuploid or polyploid DNA content. Determination of DNA ploidy in endometrial and other cancers has therefore become a powerful tool for assessing prognosis and the probability of survival in relation to recurrence of the disease, and is a better predictor than molecular or genetic markers (Lukes et al, 1994).
It is now clear that in many malignancies there is a high correlation between tumour aggressiveness and expression of proteases in the tumour tissue. In the last seven years, for example, convincing evidence has accumulated that directly implicates members of the matrix metalloproteinase (MMP) family and tissue inhibitors of metalloproteinases (TIMPs) in tumour invasion and metastasis (reviewed by Stetler-Stevenson et al, 1993). It has been suggested that detection of MMPs in biological fluids, especially as complexes with TZMPs, can be used to detect metastatic cancer (U.S. Pat. No 5,324,634 by Zucker). These enzymes are also synthesised and secreted by normal cells during physiological tissue remodelling (menstruation) and invasion (embryo implantation) (Salamonsen, 1995, 1996). MMPs may be present in tissues either in the fully active form, or in a latent form which requires activation before enzymic activity can be demonstrated.
We have recently established that MMPs are released into the uterine cavity during the ovarian cycle and early pregnancy in marmoset monkeys (our unpublished results). Moreover, over-expression of these enzymes has been reported to occur in many tumour cells, for example those of colorectal and squamous cell carcinomas.
Recently it has been demonstrated that ovarian cancer cells, but not normal ovarian epithelial cells, secrete enhanced levels of matrix metalloproteinases similar to MMP-2 and MMP-9, which directly degrade basement membrane proteins (Moser et al, 1994; Salamonsen, 1996). This proteolytic capability was correlated with the invasive potential of the ovarian malignancies. Furthermore, immunohistochemistry employing monoclonal antibodies has been used to localize MMP-2 to the cytoplasm and plasma membranes of various neoplastic cells, including endometrial cancers, whereas TIMP-2 was localized mainly to the stroma of these tissues (Hoyhtya et al, 1994). Apart from this, little is known about the expression of HMPs and TIMPs in endometrial cancers. Such information is essential for developing strategies for controlling the biological behaviour of endometrial and other types of malignancies.
We have now unexpectedly discovered that matrix metalloproteinases can be detected in uterine washings from patients with endometrial cancer, but not in similar washings from control women. This finding makes possible a sensitive diagnostic test which can be performed with minimal invasion, and a screening test which may be utilised for screening at-risk populations or the general population, or for use in clinical trials of putative treatments. The test is also suitable for monitoring the efficacy of treatment and for detection of recurrences.
In one aspect, the invention provides a method of assaying for the presence and/or risk of endometrial cancer, comprising the steps of obtaining a sample of uterine washings from a subject at risk of, or suspected to be suffering from, endometrial cancer, and measuring the levels of matrix metalloproteinases (MMPs) in the washings.
For the purposes of this specification, the expression xe2x80x9cdetection of endometrial cancerxe2x80x9d is used in its broadest sense, and includes the determination of the likely presence or absence of cancer, or cells in a precancerous state or the early phases of development of cancer. Thus the invention encompasses not only the detection of active endometrial cancer, but also the risk of developing such active cancer.
The uterine washings may be obtained by using any suitable solution which is delivered to the uterine cavity and then recovered and collected for testing. Suitable solutions will be readily apparent to those skilled in the art and include any sterile non-toxic solution which will not interfere with measurement of MMPs. An example of a suitable solution is sterile saline. Any number of means for delivery and recovery of the washing solution may be utilised, by way of example a syringe and tube are conveniently used. The washing may be collected in the recovery means or transferred to another container. Delivery, recovery and collection means will routinely be chosen to provide comfort and convenience to both the patient and clinician.
Any suitable technique for determining levels of MMPs in the washings may be employed in the method of the invention. Examples of suitable techniques include those based on determining enzyme activity, and/or those based on determining the presence or levels of enzyme. Total MMPs, including latent, active, and TIMP-bound MMP, may be measured. Those skilled in the art will readily be able to determine whether or not a technique is suitable, if necessary using positive and negative control samples. For example, gelatin zymography is suitable for detection of all forms of MMP-2 and MMP-9. Optionally the zymographic assay may be coupled with densitometry in order to provide a semi-quantitative assessment of each enzyme type and form (ie. latent or active). Active enzyme may also be assayed using substrate-based activity assays.
Total gelatinolytic activity present in the uterine washings may be measured; although this does not distinguish between the different types of MMPs present, it does produce a quick estimate of total MMP activity. However, preferably the level of activity of MMP-2 and/or MMP-9 is measured.
Alternatively total enzyme levels may be measured using methods such as ELISA, fluorometric assay, chemiluminescent assay, or radioimmunoassay. BLISA or chemiluminescent assay methods are particularly preferred, since these are quick, sensitive, and specific, and are readily automated for large scale use. These methods also provide quantitative determinations. A number of appropriate methods for measuring these enzymes are detailed in Barrett (1995).
Optionally levels of tissue inhibitors of metalloproteinases (TINPs) may also be determined in the uterine washings, for example by reverse zymography or ELISA.
In an additional embodiment of the invention, a sample of superficial endometrial cells is collected at the same time as the uterine washings are obtained, for example by insertion of a uterine brush into the uterine cavity, the endometrial cells are subjected to cytological examination for determination of ploidy and nuclear morphometry, and levels of MMPs, determined as described above, are correlated with DNA ploidy as an indicator of the prognosis of endometrial cancer when endometrial cancer is present.
Standard image cytometry techniques and automated equipment may be used for determination of ploidy and nuclear morphology.
In a second aspect, the invention provides a method for assessing the prognosis of endometrial cancer, comprising the steps of measuring MMP activity in uterine washings obtained prior to surgery in a patient undergoing surgery for endometrial cancer, obtaining a sample of endometrial cells from said patient, obtaining a sample of tumour and adjacent normal endometrium for histological examination, and correlating MAP activity, DNA ploidy and grade and type of tumour as an indicator of the prognosis.
If surgery to remove the uterus is not possible for any reason, primary treatment of endometrial cancer may be conservative, using radiotherapy, chemotherapy, endometrial ablation (for example, using photo dynamic therapy, or curettage, rather than hysterectomy); in some cases curettage is used in conjunction with other methods. In patients undergoing such conservative treatment, follow-up and detection of recurrence of cancer is particularly important.
Thus in a third aspect, the invention provides a method of monitoring the efficacy of treatment of endometrial cancer, comprising the steps of obtaining a sample of uterine washings from a patient undergoing such treatment, and measuring the levels of MMPs in the washings. This method is also applicable to monitoring efficacy of putative treatments.
In a fourth aspect, the invention provides a method of detecting recurrence of endometrial cancer in a patient who has undergone conservative treatment for primary endometrial cancer, comprising the steps of obtaining a sample of uterine washings from the patient, and measuring the levels of MKPs in the washings.
Preferably the activities of MMP-2 and MMP-9 are determined by quantitative densitometric zymography or ELISA, as described above.
The method of the invention is suitable for screening of asymptomatic or well women, or of at-risk populations, such as diabetic women or women undergoing post-menopausal hormone replacement therapy.
In a fifth aspect, the invention provides a kit for assaying the presence and/or risk of endometrial cancer, comprising:
(a) a suitable solution for washing the uterine cavity, and
(b) a means for delivery, recovery and collection of the uterine washings.
Preferably the kit further comprises:
(c) a means for determining the level of MMPs in said washings.
The kit may further optionally include one or more of:
(d) a means for taking a sample of endometrial epithelium,
(e) one or more microscope slides, and
(f) a receptacle containing a histological fixative.
In one preferred embodiment, the kit comprises:
(a) a sterile physiologically-acceptable solution for washing the uterine cavity,
(b) a sterile syringe,
(c) a sterile feeding tube or catheter,
(d) a receptacle for uterine washings, and optionally,
(e) a uterine brush, and/or
(f) one or more microscope slides.
Preferably the kit additionally comprises a receptacle containing a histological fixative; more preferably the fixative is 4% paraformaldehyde.
For the purposes of this specification it will be learly understood that the word xe2x80x9ccomprisingxe2x80x9d means xe2x80x9cincluding but not limited toxe2x80x9d, and that the word xe2x80x9ccomprisesxe2x80x9d has a corresponding meaning.